Therapy and prevention of prion protein complex infections

ABSTRACT

There are disclosed therapies and preventions of prion protein complex infections. The transcription of the amyloid precursor protein gene and PrP gene and the RNA transcript are the rate-limiting steps and are most susceptible for blockage and control of the process of amyloid protein formation and PrPsc formation. Thus, therapies and prevention regimes for prion protein complex infections interrupt this process at the level of DNA transcription to RNA, RNA transport to the mitochondrion for protein synthesis and deposition in the cerebral cortex neurons.

RELATED APPLICATION INFORMATION

This application is a division of application Ser. No. 16/118,349 filedAug. 30, 2018 entitled “Therapy and Prevention of Prion Protein ComplexInfections” which claims priority from the following provisional patentapplications:

Application No. 62/690,736 filed Jun. 27, 2018 entitled “Treatment ofAlzheimer's Disease;” Application No. 62/691,910 filed Jun. 29, 2018entitled “Preventive Therapy of Alzheimer's Disease;” and ApplicationNo. 62/714,012 filed Aug. 2, 2018 entitled “Therapy and Prevention ofAlzheimer's Disease.”

A portion of the disclosure of this patent document contains materialwhich is subject to copyright protection. This patent document may showand/or describe matter which is or may become trade dress of the owner.The copyright and trade dress owner has no objection to the facsimilereproduction by anyone of the patent disclosure as it appears in thePatent and Trademark Office patent files or records, but otherwisereserves all copyright and trade dress rights whatsoever.

BACKGROUND Field

This disclosure relates to therapy and prevention of prion proteincomplex infections.

Description of the Related Art

AD is commonly believed to be a localized brain disease. AD withneurological disease is the third leading cause of death in the UnitedStates after cardiovascular diseases and cancer. AD normally follows asequence comprised of neuro-inflammation, amyloid and tau proteopathy,accumulative storage disease, neurotoxicity and neurodamage, loss offunction (i.e., activities of daily living (ADL) and cognitive skills),and finally death. AD deaths are due to the futility and loss of will tolive in these patients who have been depersonalized and lost the will tolive, coupled with the failure to thrive leading to premature deathusually within five to ten years of diagnosis of AD.

There are four main prevailing theories about the causation of AD: (a) acholinergic hypothesis, (b) an amyloid protein deposition hypothesis,(c) a tau protein deposition hypothesis, and (d) a neurovascularhypothesis. Presently there is no effective treatment capable ofmodifying the progression of Alzheimer's disease, or preventing itsonset. Currently available therapies only act on symptomaticimprovement, while the development of therapies capable of blocking ordelaying the disease progression remains a challenging unmet need.

According to the cholinergic hypothesis, degeneration of cholinergicneurons in the basal forebrain and the associated loss of cholinergicneurotransmission in the cerebral cortex and other areas contributedsignificantly to the deterioration in cognitive function seen inpatients with Alzheimer's disease.

Under the amyloid protein deposition hypothesis, the formation ofamyloid plaques and neurofibrillary tangles are thought to contribute tothe degradation of the neurons (nerve cells) in the brain and thesubsequent symptoms of Alzheimer's disease. Amyloid proteins are a largegroup of proteins of which sixty different types have been described.Thirty-six amyloid proteins have been associated with human disease.Amyloid protein was first seen and described by Rudolf Virchow whothought it was a starchy substance hence the name amyloid related tostarch or “amylin” in Latin. It was next thought to be a fattysubstance, but later found to be a protein substance. Since theintroduction of elegant protein chemistry, mass spectrometry, and x-raycrystallography, amyloid proteins have been better characterized andclassified in various human diseases and conditions.

Amyloid protein disease was once classified as primary or secondary.Primary disease was recognized as synthesis and deposition of theprotein in organs such as the heart, kidney, skin, tongue etc. Insecondary disease, amyloid protein deposition was recognized assecondary to a chronic suppurative condition such as tuberculosis orother uncontrolled bacterial abscess which is common in developingnations of the world. Similarly, chronic inflammatory conditions, suchas rheumatoid arthritis and renal dialysis, lead to reactive amyloidprotein deposition.

Amyloid precursor protein (APP), which is encoded in chromosome 21, hasa role in AD. APP is a trans-membrane protein that penetrates throughthe neuron's membrane, and is critical for neuron growth, survival, andpost-injury repair. Thus, loss of a neuron's APP may affectphysiological deficits that contribute to dementia. Clinical data fromindividuals with Down syndrome (i.e., trisomy 21) shows that theydevelop AD earlier in their 40 s, since the gene for APP is inchromosome 21, and they are saddled with three copies. This is akin topatients with inflammatory bowel disease (IBD) who develop colon cancerin their 30-40 s compared to normal population who develop it in their50 s to 80 s. APP is copied and used to synthesize amyloid protein.

Amyloid beta (Aß) is the specific amyloid protein implicated in AD.Amyloid plaques are made up of small peptides, 39-43 amino acids inlength. Amyloid beta is produced from the sequential cleavage of APP bybeta-site amyloid precursor protein-cleaving enzyme 1 (BACE-1) followedby gamma-secretase. In AD, gamma secretase and beta secretase acttogether in a proteolysis catabolic reaction, cleaving a smallerfragment of APP. These protein catabolism fragments then form fibrils ofamyloid beta, which further form clumps deposited outside the neuronsknown as senile plaques.

Because Aβ accumulates excessively in AD, there is a logical inferencethat its precursor, APP, would be elevated as well. However, a study hasshown that neuronal cell bodies contain less APP as a function of theirproximity to amyloid plaques. It has been theorized that this APPdeficit near Aß plaques results from a decline in production of APPwhich normally rises in response to stress.

Several BACE-1 inhibitors and humanized monoclonal antibodies to solubleamyloid protein have been in clinical trials in AD. These trials failedto deliver on the promise of being disease modifying drug (DMD) agents(i.e., they change the underlying pathology of the disease) in AD.Similarly, vaccines have been tried to clear amyloid protein plaques inAD all to no avail. In light of the failure of clinical trials usingBACE inhibitors, and the failure of amyloid immunotherapy withintravenous Solanezumab, the amyloid protein deposition theory has beencalled into question.

The tau protein deposition hypothesis proposes that tau proteinabnormalities initiate the disease cascade. In this model,hyperphosphorylated tau begins to pair with other threads of tau.Eventually, they form neurofibrillary tangles inside nerve cell bodiesWhen this occurs, the microtubules disintegrate, destroying thestructure of the cell's cytoskeleton which collapses the neuron'stransport system. This may result first in malfunctions in biochemicalcommunication between neurons and later in the death of the cells.

The neurovascular hypothesis claims that a substantial amount of Aßpeptide in the brain of Alzheimer's disease patients is originated inthe systemic circulation. According to this theory, poor functioning ofthe blood—brain barrier (BBB) is involved. One side effect of this poorfunction is production of amyloid and tau hyper-phosphorylation.

Prion (PrP) is a protein which arises from misfolding of a normalprotein. The two forms of prion are designated as PrP^(c), which is anormally folded protein, and PrP^(sc), a misfolded form which gives riseto the disease. The two forms do not differ in their amino acidsequence, however the pathogenic PrP^(sc) isoform differs from thenormal PrP^(c) form in its secondary and tertiary structure. ThePrP^(sc) isoform is more enriched in beta sheets, while the normalPrP^(c) form is enriched in alpha helices. The differences inconformation allow PrP^(sc) to aggregate and be extremely resistant toprotein degradation by enzymes or by other chemical and physical means.The normal form, on the other hand, is susceptible to completeproteolysis and soluble in non-denaturing detergents. It has beensuggested that pre-existing or acquired PrP^(sc) can promote theconversion of PrP^(c) into PrP^(sc), which goes on to convert otherPrP^(c). This initiates a chain reaction that allows for its rapidpropagation, resulting in the pathogenesis of prion diseases. PrP^(c)protein is one of several cellular receptors of soluble amyloid beta(Aβ) oligomers.

Against this background of prion protein complex infections, we turn toseveral drugs which have not been proposed for therapeutic applicationtoward prion protein complex infections. For example, althoughgenetically engineered antibodies have been tried, antibiotics have notbeen considered as possible therapies for prion protein complexinfections. Another class not previously considered areimmunosuppressants.

The tetracyclines are a very old group of bacteriostatic antibioticsconsisting of tetracycline, doxycycline and minocycline. They act byinhibiting protein synthesis in bacterial and protozoa cells and ineukaryotic organism mitochondrion, inhibiting the binding ofaminoacyl-tRNA to the mRNA ribosome complex. They do so mainly bybinding to the 30S ribosomal subunit in the mRNA translation complex. Inaddition to inhibiting protein synthesis, these drugs areanti-inflammatory, are lipid soluble, and have high central nervoussystem concentration.

Sirolimus, also known as rapamycin, is a macrolide compound marketedunder the trade name Rapamune by Pfizer. Sirolimus has immunosuppressanteffects in humans and is used in preventing the rejection of kidneytransplants. It inhibits activation of T cells and B cells by reducingtheir sensitivity to interleukin-2 (IL-2) through mTOR inhibition. Byits effect on B cells it prevents the humoral immune system fromsynthesizing humoral antibodies to the renal graft.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a skeletal formula of tetracycline with atoms and four ringsnumbered and labeled.

FIG. 2 is a formula for doxycycline.

FIG. 3 is a formula for minocycline.

FIG. 4 is a formula for sirolimus.

FIG. 5 is a conceptual diagram showing how the basis for exponentialgrowth of the presence of Aβ and PrP^(sc).

DETAILED DESCRIPTION

AD is not a localized brain disease. Like other prion protein complexinfections, AD is a systemic disease involving both the body and theperipheral circulation and B-cells. AD includes a localized reaction inthe neocortex. Indeed, proof of this is the fact that AD can bediagnosed in saliva by testing for Ab42 level (with ELISA test), bloodAb42/40 ratio, and cerebrospinal Ab42 level.

Amyloid beta protein deposition seen in AD is secondary to a chronicneuro-inflammatory condition in the acetylcholine discharging neurons ofthe cerebral cortex. This amyloid protein deposition starts ten tofifteen years prior to the clinical diagnosis of AD in the patient andcontinues until the patient dies. The transcription of the APP gene andthe RNA transcript are the rate-limiting steps and are most susceptiblefor blockage and control of the process of amyloid protein formation.Thus, there is described herein a cure for AD based upon interruption ofthis process at the level of DNA transcription to RNA, RNA transport tothe mitochondrion for protein synthesis and deposition in the cerebralcortex neurons. This is the main thrust of our effort in introducing thefirst DMDs in AD.

This neuroinflammation in the neocortex is concomitant with localizedsecretion of amyloid beta to the acetylcholine secreting memory nervefibers, and the secretion of cellular prion protein (PrP^(c)) peptidesand tau protein peptides. Because of the neurotoxicity of the amyloidprotein oligomers there is the misfolding of the PrP^(c) peptidesconverting them from an alpha helical structure to a beta helicalstructure (i.e., PrP^(sc)). The PrP^(sc) beta helical structureinteracts with Aß fibrils and starts laying down sheets of Aß fibrilswhich are neurotoxic and lead to neurotoxicity and nerve fiber and nervecell death creating the pathognomonic amyloid plaques and the tauprotein tangles.

The preventative and curative therapies described herein for prionprotein complex infections have a dual interaction, and this dualinteraction is necessary to halt the progress of the disease, undo atleast some of its damage, and prevent it from re-arising or recurring.FIG. 5 demonstrates the basis for exponential growth of the presence ofAß and PrP^(sc). As shown in FIG. 5, when PrP^(sc) is applied toPrP^(c), the PrP^(c) misfolds into PrP^(sc). The same behavior ariseswith APP and Aβ: Aß is a seed for producing more Aß from APP. However,these two cycles are not independent. They are interdependent. That is,Aß seeds conversion of PrP^(c) into PrP^(sc), and PrP^(sc) seedsproduction of Aß from APP. Thus, this witch's brew of Aß and PrP^(sc) atthe heart of prion protein complex infections is a cycle of death whichcannot be stopped by a therapy which only interferes with misfolding ofPrP^(c) into PrP^(sc), or only interferes with production of Aß fromAPP. The therapy described herein addresses both types of misfolding.

Prion protein complex infections may be treated and prevented throughtwo treatment forms. In these infections, amyloid beta protein, presentin the blood, diffuses into the cerebrospinal fluid which washes overthe brain and the neocortex. This creates a secondary neocorticalreaction with the laying of sheets and sheets of amyloid beta fibrils,leading to the death and destruction of memory cells and creatingamyloid plaques and neurofibrillary tau protein tangles. One treatmentform uses an immunosuppressant to address the systemic humoral B cellreaction and prion protein transcription, translation and synthesis. Theother treatment form uses an antibiotic to address synthesis of amyloidbeta protein. Benefits are obtained by combining the treatment forms.

Prion protein complex infections arise from a complex of rogue prionproteins—a “witches brew.” This rogue prion protein complex consists ofAß fibrils and prion protein beta (PrP^(sc)) fibrils. The body's naturalreaction to the rogue prion protein complex is a self-defense mechanismthat itself irreparably destroys tissue. These defense mechanisms takethe form of a self-assembling Pacman which attacks and eats the rogueprion protein complex. Injury to the corresponding tissue is the culpritin the pathogenesis of AD and other prion protein complex infections.

The systemic disease component of prion protein complex infections maybe treated with an immunosuppressant such as sirolimus. Sirolimus, byits effect on B cells, impairs the humoral immune system fromsynthesizing humoral antibodies and APP. This abrogates the systemiccomponent of the AD pathogenesis.

The central nervous system (CNS) localized effects of prion proteincomplex infections may be treated with antibiotics such astetracyclines. Tetracyclines block protein synthesis by their effects ontranscription, translation, and binding to the ribosomal proteincomplexes. The tetracycline compounds can deal with the CNS/neocorticalcomponent of the AD pathogenesis by inhibiting the transcription of theAPP gene on chromosome 21 and the transcription of the PrP gene onchromosome 20. Additionally, the tetracyclines block translation of thegene and protein synthesis by binding to the 30S and 50S subunits of theribosomal protein complex.

The double action through treatment of both the systemic diseasecomponent and the CNS localized effects leads to hindering or abolitionof the effect of the rogue prion protein complex. By inhibiting thetranscription and blocking the synthesis of amyloid protein in ADpatients, we stop further amyloid protein deposition in the cerebralcortex and the subsequent neurotoxicity and neuronal damage and loss ofmemory and function. Patients accordingly regain function and are ableto participate in their activities of daily living and interactions withfamily members. Similarly, by blocking the transcription and synthesisof PrP^(sc), the second part of the rogue prion protein complex isdisrupted.

For patients with AD, the therapy may be either an antibiotic alone, oran antibiotic in combination with an immunosuppressant. For an adult, anappropriate therapy may be one of the following: (a) doxycycline 100 mgtwice per day such as in the morning and in the evening; (b) a firstdose of doxycycline 100 mg and sirolimus 2 mg taken together, such as inthe morning, and a separate dose of doxycycline 100 mg at another time,such as in the evening; (c) minocycline 100 mg and sirolimus 2 mg takenat the same time such as in the morning; (d) a single dose ofminocycline 100 mg, such as in the morning.

A dose may take the form on a unit dose. That is, a unit dose is a pill,a tablet or a capsule—one and only one.

Effectiveness of this therapy is apparent in three to twelve months.Once treatment is effective, the patient may discontinue the therapyunder controlled observation for relapse and possible retreatment. ForAD patients, effectiveness may be measured by the Alzheimer's DiseaseAssessment Scale-Cognitive (ADAS-Cog) subscale and the Alzheimer'sDisease Cooperative Study-Activities of Daily Life (ADCS-ADL) scale.Both of these tests have been developed over many years, and it isexpected that they will continue to be refined.

These therapies may be varied in a number of ways. First, otherinhibitors of protein synthesis at the level of transcription,translation and protein assembly may be used. Second, the dosage levelsmay be different, with daily dosages of doxycycline as low as 40 mg,minocycline as low as 25 mg, and sirolimus as low as 0.5 mg. On theupper end the dosages may be as much as 400 mg (e.g., 200 mg twice perday) of doxycicline, 300 mg (e.g., 150 mg twice per day) of minocycline,and 4 mg (e.g., 2 mg twice per day) of sirolimus. The dosages specifiedabove are for an average adult, and dosage may be correlated to bodyweight, with heavier patients receiving a larger dose and lighterpatients receiving a smaller dose. Dosages need not be correlated toage. Dosages may be slow release.

How often the pill is taken may be varied, as may the time of day. Everyother day may be sufficient for some patients, or three days on and twodays off. These are examples of drug holidays. Dosage may be differentday-to-day. Time of day for taking the medication may be selected basedupon the patient having an empty stomach for better absorption.

For individuals with a first or second degree relative diagnosed with ADand a positive saliva amyloid beta 42 test of 40 pg/ml or greater byELISA, the same regime prevents AD or effectively treats undiagnosed ADand may be used as a preventative therapy.

Other antibiotics which may be used that inhibit protein genetranscription, translation and synthesis.

Other immunosuppressants may be used that block B cell function andsynthesis of amyloid beta and PrP^(sc), such as are cyclosporin,tacrolimus and everolimus.

Despite the failure of BACE inhibitors in treating AD, the amyloidprotein deposition hypothesis is valid. These studies failed because theinhibitors acted downstream in the metabolism of amyloid protein. Thetherapies described herein work at the level of DNA transcription to RNAand RNA transport to the mitochondrion for protein synthesis by bindingto the 30S and 50S subunits of the RNA to block amyloid proteinsynthesis. Plus, normal cellular catabolism eliminates already depositedamyloid protein.

The therapies described above are also effective for other prion proteincomplex infections. These include Creutzfeldt Jakob disease (CJD), Lewybody disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS),cerebral amyloid angiopathy, and Down's syndrome.

Closing Comments

Throughout this description, the embodiments and examples shown shouldbe considered as exemplars, rather than limitations on the apparatus andprocedures disclosed or claimed. Although many of the examples presentedherein involve specific combinations of method acts or system elements,it should be understood that those acts and those elements may becombined in other ways to accomplish the same objectives. With regard toflowcharts, additional and fewer steps may be taken, and the steps asshown may be combined or further refined to achieve the methodsdescribed herein. Acts, elements and features discussed only inconnection with one embodiment are not intended to be excluded from asimilar role in other embodiments.

As used herein, “plurality” means two or more. As used herein, a “set”of items may include one or more of such items. As used herein, whetherin the written description or the claims, the terms “comprising”,“including”, “carrying”, “having”, “containing”, “involving”, and thelike are to be understood to be open-ended, i.e., to mean including butnot limited to. Only the transitional phrases “consisting of” and“consisting essentially of”, respectively, are closed or semi-closedtransitional phrases with respect to claims. Use of ordinal terms suchas “first”, “second”, “third”, etc., in the claims to modify a claimelement does not by itself connote any priority, precedence, or order ofone claim element over another or the temporal order in which acts of amethod are performed, but are used merely as labels to distinguish oneclaim element having a certain name from another element having a samename (but for use of the ordinal term) to distinguish the claimelements. As used herein, “and/or” means that the listed items arealternatives, but the alternatives also include any combination of thelisted items.

1. A method of treating an individual having a prion protein complexinfection which is Alzheimer's disease or preventing an individual fromhaving a prion protein complex infection which is Alzheimer's disease,the infection characterized by amyloid beta pathology and PrP^(sc)fusion protein pathology, comprising: administering to the individual aneffective amount of a medication comprising an antibiotic which isminocycline, wherein the medication interrupts transcription of a genefor amyloid precursor protein and RNA transcript at the level of DNAtranscription to RNA, RNA transport to the mitochondrion for proteinsynthesis and deposition in the cerebral cortex neurons; andadministering to the individual an effective amount of animmunosuppressant which is sirolimus, wherein the immunosuppressantimpairs the humoral immune system from synthesizing humoral antibodies,amyloid beta and PrP^(sc) proteins. 2-8. (canceled)
 9. The method ofclaim 1 wherein administering sirolimus comprises 2 mg of sirolimus onceper day.
 10. The method of claim 1 wherein the antibiotic and theimmunosuppressant are administered together as a unit dose. 11.(canceled)
 12. The method of claim 1 wherein administering an effectiveamount of the antibiotic and administering an effective amount of theimmunosuppressant comprises administering the antibiotic and theimmunosuppressant for at least three months.
 13. A method of reducingamyloid beta protein and PrP^(sc) fusion protein complex in a patientwith Alzheimer's disease comprising: administering to the patient aneffective amount of an antibiotic, wherein the antibiotic comprises atetracycline which is minocycline, wherein the antibiotic interruptstranscription of a gene for amyloid precursor protein and PrP^(sc)fusion protein complex and the RNA transcript at the level of DNAtranscription to RNA, RNA transport to the mitochondrion for proteinsynthesis and deposition in the cerebral cortex neurons; administeringto the patient an effective amount of an immunosuppressant which issirolimus, wherein the immunosuppressant impairs the humoral immunesystem from synthesizing humoral antibodies, amyloid beta and PrPscproteins. 14-15. (canceled)
 16. The method of claim 13 whereinadministering the antibiotic comprises 50 mg twice per day. 17.(canceled)
 18. The method of claim 13 wherein administering theantibiotic comprises 100 mg of minocycline once per day.
 19. (canceled)20. (canceled)
 21. The method of claim 13 wherein administering theimmunosuppres sant comprises 2 mg of sirolimus once per day.
 22. Themethod of claim 13 wherein the tetracycline and the immunosuppressantare administered together as a unit dose.
 23. (canceled)
 24. The methodof claim 13 wherein administering an effective amount of the antibioticand administering an effective amount of the immunosuppressant comprisesadministering the antibiotic and the immunosuppressant for at leastthree months.
 25. (canceled)
 26. A method of treating an individualhaving a prion protein complex infection which is Alzheimer's disease orpreventing an individual from having a prion protein complex infectionwhich is Alzheimer's disease, consisting essentially of: administeringto the individual an antibiotic or a pharmaceutically acceptable saltthereof, wherein the antibiotic comprises a minocycline; animmunosuppressive or a pharmaceutically acceptable salt thereof, whereinthe immunosuppressive comprises sirolimus; and one or morepharmaceutically acceptable carriers, diluents, or excipients.
 27. Themethod of claim 26 wherein the minocycline is administered in a dose of50 mg-100 mg.
 28. (canceled)
 29. The method of claim 26 whereinsirolimus is administered in a dose of 0.5 mg-2 mg.
 30. The method ofclam 26 wherein administration of the minocycline and the sirolimusoccurs at least twice per day.